Biomedical subjects
Publications and source records attributed to R Padmakumar.
A rare combination of tetralogy of Fallot, absent pulmonary valve and absent left pulmonary artery is described. The clinical and angiographic profile and the impact on management are discussed. The embryology is outlined.
A young lady with post-subclavian coarctation and cranial hypertension is discussed. She had a jump graft from left subclavian artery to descending aorta, and presented 18 years later with hypertension, calcification of the graft with a gradient of 40 mmHg across it. In the meantime, she also developed moderate aortic regurgitation on a bicuspid aortic valve. Management strategies including trans-catheter options are discussed. We present our reservations on trans-catheter options in an occluded dacron graft.
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Expression of nitrile hydratase enzymes utilized in a new "green" process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobalt ion into this host. We describe the expression of a thermostable nitrile hydratase from a moderate thermophile Bacillus sp. BR449 in E. coliin which the cobalt required for enzyme activation is introduced by incubation of the apoenzyme in the presence of Co++ ion at 50 degrees C, yielding active and thermostable enzyme.
A case of foreign body (denture) in upper gastro-intestinal tract in an adult male is reported because of its uncommon occurrence, and failure to remove it by laryngoscopy or oesophagogastroscopy. The peculiar nature of the denture and the accessories necessitated laparotomy-gastrotomy.
Taurine/alpha-ketoglutarate dioxygenase (TauD), a member of the broad class of non-heme Fe(II) oxygenases, converts taurine (2-aminoethanesulfonate) to sulfite and aminoacetaldehyde while decomposing alpha-ketoglutarate (alphaKG) to form succinate and CO(2). Under anaerobic conditions, the addition of alphaKG to Fe(II)TauD results in the formation of a broad absorption centered at 530 nm. On the basis of studies of other members of the alphaKG-dependent dioxygenase superfamily, we attribute this spectrum to metal chelation by the substrate C-1 carboxylate and C-2 carbonyl groups. Subsequent addition of taurine perturbs the spectrum to yield a 28% greater intensity, an absorption maximum at 520 nm, and distinct shoulders at 480 and 570 nm. This spectral change is specific to taurine and does not occur when 2-aminoethylphosphonate or N-phenyltaurine is added. Titration studies demonstrate that each TauD subunit binds a single molecule of Fe(II), alphaKG, and taurine. In addition, these studies indicate that the affinity of TauD for alphaKG is enhanced by the presence of taurine. alpha-Ketoadipate, the other alpha-keto acid previously shown to support TauD activity, and alpha-ketocaproate lead to the formation of weak 520 nm-like spectra with Fe(II)TauD in the presence of taurine; however, corresponding spectra at 530 nm are not observed in the absence of taurine. Pyruvate and alpha-ketoisovalerate fail to elicit absorption bands in this region of the spectrum, even in the presence of taurine. Stopped-flow UV-visible spectroscopy reveals that the 530 and 520 nm spectra associated with alphaKG-Fe(II)TauD and taurine-alphaKG-Fe(II)TauD are formed at catalytically competent rates ( approximately 40 s(-)(1)). The rate of chromophore formation was independent of substrate or enzyme concentration, suggesting that alphaKG binds to Fe(II)TauD prior to the formation of a chromophoric species. Significantly, the taurine-alphaKG-Fe(II)TauD state, but not the alphaKG-Fe(II)TauD species, reacts rapidly with oxygen (42 +/- 9 s(-)(1)). Using the data described herein, we develop a preliminary kinetic model for TauD catalysis.
Although providing an attractive route for production of acrylamide from acrylonitrile, utilization of nitrile hydratase enzymes has been limited by the requirement for low temperature bioconversion conditions. This report summarizes a search for thermostable nitrile hydratases from aerobic moderate thermophiles screened for ability to grow on acrylonitrile at concentrations to 1% at elevated temperatures. A new isolate Bacillus sp. BR449 constitutively expresses a thermostable nitrile hydratase with properties including low substrate inhibition and broad temperature range with optimal activity at 55 degrees C. With prolonged exposure, BR449 nitrile hydratase exhibited temperature- dependent inactivation by acrylonitrile, which is attributed to alkylation of nucleophilic sites on the enzyme.
Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. It is dependent on the cofactor, coenzyme B12 or adenosylcobalamin, for activity. The first step in this, and other coenzyme B12-dependent reactions, is postulated to be homolysis of the Co-C bond of the cofactor. Methylmalonyl-CoA mutase accelerates the rate of Co-C bond homolysis by a factor of approximately 10(12). The strategy employed by the enzyme for the remarkable labilization of this bond is not known. Using UV-visible stopped-flow spectrophotometry, we demonstrate that the Co-C homolysis rate in the presence of protiated substrate has a rate constant of >600 s(-1) at 25 degrees C. In the presence of [CD3]methylmalonyl-CoA, this rate decreases to 28 +/- 2 s(-1). These results suggest that Co-C bond homolysis is coupled to hydrogen atom abstraction from the substrate and that the intrinsic binding energy of substrate may be a significant contributor to catalysis by methylmalonyl-CoA mutase.
Enzymes with radical-pair intermediates have been considered as a likely target for purported magnetic field effects in humans. The bacterial enzyme ethanolamine ammonia lyase and the human enzyme methylmalonyl-CoA mutase catalyze coenzyme B12-dependent rearrangement reactions. A common step in the mechanism of these two enzymes is postulated to be homolysis of the cobalt-carbon bond of the cofactor to generate a spin-correlated radical pair consisting of the 5'-deoxyadenosyl radical and cob(II)alamin [Ado. Cbl(II)]. Thus, the reactions catalyzed by these enzymes are expected to be sensitive to an applied magnetic field according to the same principles that control radical pair chemical reactions. The magnetic field effect on ethanolamine ammonia lyase reported previously has been corroborated independently in one of the authors' laboratory. However, neither the human nor the bacterial mutase from Propionibacterium shermanii exhibits a magnetic field effect that could be greater than about 15%, considering the error limit imposed by the uncertainty of the coupled assay. Our studies suggest that putative magnetic field effects on physiological processes are not likely to be mediated by methylmalonyl-CoA mutase.
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Recombinant methylmalonyl-coenzyme A (CoA) mutase from Propionibacterium shermanii has been purified 20-fold to near homogeneity in a highly active form. Neither the apoenzyme (the form in which the enzyme is isolated) nor the holoenzyme (reconstituted with the cofactor, adenosylcobalamin) has an electron paramagnetic resonance (EPR) spectrum associated with it. However, the addition of either the substrate, methylmalonyl-CoA, or the product, succinyl-CoA, results in the appearance of a transient EPR signal. The signal has hyperfine features that indicate coupling of the unpaired electron to the cobalt nucleus. In the presence of [CD3]methylmalonyl-CoA, an EPR signal is also seen and is similar to that obtained in the presence of protiated substrate. Power saturation studies reveal the presence of two components, a slow relaxing species (with an apparent g value of 2.11) and a fast relaxing species (with an apparent g value of 2.14) that can be partially resolved at low temperature and high power. The EPR-active intermediate is observed under catalytic conditions and is approximately midway in its resonance position between a free radical and cob(II)alamin. It is postulated to represent an exchange-coupled cob(II)alamin ... free radical pair. The signal bears close resemblance to those observed with partially dehydrated polycrystalline adenosylcobalamin following laser photolysis (Ghanekar, V.D., Lin, R.J., Coffman, R.E., and Blakley, R.L. (1981) Biochem. Biophys. Res. Commun. 101, 215-221) and with the adenosylcobalamin-dependent ribonucleotide reductase under freeze-quench conditions (Orme-Johnson, W.H., Beinert, H., and Blakley, R.L. (1974) J. Biol. Chem. 249, 2338-2343). When cob(II)alamin is generated under noncatalytic conditions (i.e. in the presence of propionyl-CoA or by electrochemical reduction of enzyme-bound hydroxocob-(III)alamin), a different EPR signal is observed with g = 2.26 and g = 2.00, typical of base-on cob(II)alamin.
Human methylmalonyl-CoA mutase is inhibited by ethylmalonyl-CoA, cyclopropylcarbonyl-CoA carboxylate, and methylenecyclopropylacetyl-CoA, which are substrate, intermediate, and product analogs, respectively. The mode of inhibition by each analog is reversible and mixed with respect to the substrate, methylmalonyl-CoA. This implies that the inhibitors are able to bind to both free enzyme and to the enzyme-substrate complex, although with affinities that are 4.5- to 10-fold different for the two species. The Ki1 for the cyclopropylcarbonyl-CoA carboxylate (0.26 +/- 0.07 mM), is 4-fold greater than the Km(app) measured for the substrate, methylmalonyl-CoA. Additionally, ethylmalonyl-CoA functions as an alternate substrate and is metabolized to methylsuccinyl-CoA. The human mutase is a homodimer that binds 1 mol of cobalamin per subunit. So, the observed mixed inhibition kinetics by substrate analogs is curious. Our finding that methylenecyclopropylacetyl-CoA, the causative agent of Jamaican "vomiting sickness," inhibits methylmalonyl-CoA mutase, while interesting, is probably not physiologically important because of the relatively high inhibition constants (Ki1 = 0.47 +/- 0.12 mM and Ki2 = 2 +/- 0.34 mM) observed with this compound.
A rapid and high-yield synthesis of methylmalonyl coenzyme A is reported. The two-step procedure involves preparation of the thiophenyl ester of methylmalonic acid using dicyclohexylcarbodiimide as a condensing agent, followed by transesterification with coenzyme A, to yield methylmalonyl coenzyme A in 80% overall yield. Inclusion of an additional step, methylation of malonic acid with iodomethane, affords the opportunity for introducing a stable or radioactive isotope into the product. This method should be applicable for the syntheses of other coenzyme A esters that are of biochemical interest such as succinyl coenzyme A.
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BACKGROUND: There is a paucity of data regarding the long-term outcome of patients operated for ventricular septal defect with severe pulmonary arterial hypertension and elevated pulmonary vascular resistance. METHODS AND RESULTS: We evaluated the long-term follow-up results of a selected cohort of patients with nonrestrictive ventricular septal defect and elevated pulmonary vascular resistance (>6 Wood units). Thirty-eight patients, median age 7.5 years (range 6 months-27 years), with nonrestrictive ventricular septal defect with severe pulmonary hypertension were operated between 1985 and 1996 at our institute. Preoperative pulmonary vascular resistance, ratio of pulmonary blood flow to systemic blood flow, and ratio of pulmonary vascular resistance to systemic vascular resistance were 7.63+/-1.8 Wood units, 1.9+/-0.48, and 0.41+/-0.12, respectively. The majority (68.4%) had perimembranous ventricular septal defect. Thirty patients (79%) had a good outcome and were asymptomatic at a mean follow-up of 8.7 years, with significant reduction in pulmonary artery pressures. Eight patients (21%) had a poor outcome, which included 5 immediate postoperative deaths, 1 late death and 2 surviving patients with persistent severe pulmonary arterial hypertension. There was no significant difference regarding hemodynamic parameters at baseline between those who had a good outcome and those who did not. Eleven patients with a preoperative pulmonary blood flow to systemic blood flow ratio of <2:1. who had a good outcome following surgery, underwent repeat catheterization at follow-up. There was a significant reduction in their mean pulmonary vascular resistance (8.03+/-1.4 v. 4.16+/-1.6 Wood units, p=0.001) and pulmonary vascular resistance to systemic vascular resistance ratio (0.41+/-0.12 v. 0.19+/-0.06, p=0.05). CONCLUSIONS: The late results of surgery on this selected group of patients with nonrestrictive ventricular septal defect with high pulmonary vascular resistance are encouraging. Operative correction of the ventricular septal defect should be actively considered in all children presenting with nonrestrictive ventricular septal defect with a significant left-to-right shunt, despite moderately elevated pulmonary vascular resistance. Even among older patients with ventricular septal defect and moderately elevated pulmonary vascular resistance, there is a specific group that does well after operation.
Methylmalonyl-CoA mutase is a member of the coenzyme B12-dependent family of isomerases and interconverts methylmalonyl-CoA and succinyl-CoA. We have examined the ability of the enzyme to effect a double rearrangement reaction when presented with glutaryl-CoA, a substrate analog with a three-carbon template on which two successive 1,2 migrations can occur. Our results demonstrate that the enzyme converts glutaryl-CoA to both methylsuccinyl-CoA and ethylmalonyl-CoA. To our knowledge, this is the first example of a double rearrangement reaction catalyzed by a coenzyme B12-dependent enzyme.